Within the cell, important molecules called tetrahydrofolates (THF) power the life-sustaining processes of DNA synthesis, replication and repair by coenzymatically providing substrates necessary for these processes. THF are biosynthesized intracellularly through reduction of folic acid or other dihydrofolate intermediates by the enzyme dihydrofolate reductase (DHFR). The pteridine compound, methotrexate (MTX; N-[4-[[(2,4-diamino-6pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid ), is structurally quite similar to folic acid. As a result of this structural similarity, MTX can bind to active sites on DHFR, and, through competitive inhibition, block the formation of THF needed in the biosynthesis of DNA and RNA.
This ability of MTX to inhibit nucleic acid synthesis has been exploited in the treatment of aberrant cell growth. In particular, since many malignant cells proliferate more rapidly than normal cells, and since actively proliferating cells are more sensitive to the effect of MTX, in many cases, MTX can be used to selectively impair cancerous cell growth without damaging normal cell growth. As a result of its effectiveness against rapidly proliferating cells, MTX is one the most widely used anticancer agents. For example, MTX is employed in the treatment of neoplastic diseases such as gestational choriocarcinoma, chorioadenoma destruens, hydatidiform mole, acute lymphocytic leukemia, breast cancer, epidermoid cancers of the head and neck, advanced mycosis fungoides, lung cancer, and non-Hodgkins lymphomas (Physicians Desk Reference (45th ed.), Medical Economical Co., Inc., 1185-89 (Des Moines, Iowa (1991))). Moreover, MTX is an effective immunosuppressive agent, with utility in the prevention of the graft-versus-host reaction that can result from tissue transplants, as well as in the management of inflammatory diseases. Consequently, MTX can be employed in the treatment of severe and disabling psoriasis and rheumatoid arthritis (Hoffmeister, The American Journal of Medicine, 30, 69-73 (1983); Jaffe, Arthritis and Rheumatism, 31, 299 (1988)).
The numerous patents that have been issued disclosing MTX and MTX analogs, methods of synthesizing MTX or analogs thereof, and uses for MTX attest to the significance of MTX in treatment of aberrant cell growth. For example, U.S. Pat. No. 2,512,572 covers the active agent MTX, and U.S. Pat. Nos. 3,892,801, 3,989,703, 4,057,548, 4,067,867, 4,079,056, 4,080,325, 4,136,101, 4,224,446, 4,306,064, 4,374,987, 4,421,913, and 4,767,859 claim methods for preparing MTX or potential intermediates in the synthesis of MTX. Other patents disclose labelled analogs of MTX, such as U.S. Pat. Nos. 3,981,983, 4,043,759, 4,093,607, 4,279,992, 4,376,767, 4,401,592, 4,489,065, 4,622,218, 4,625,014, 4,638,045, 4,671,958, 4,699,784, 4,785,080, 4,816,395, 4,886,780, 4,918,165, 4,925,662, 4,939,240, 4,983,586, 4,997,913, 5,024,998, 5,028,697, 5,030,719, 5,057,313, 5,059,413, 5,082,928, 5,106,950, and 5,108,987, wherein MTX is bound to a radionucleotide or fluorescent label, amino acid, polypeptide, transferrin or ceruloplasmin, chondroitin or chondroitin sulfate, antibody, or binding partner for a specific cell-surface receptor of target cells for use in assays of MTX, in timed-release of MTX, as toxins selective for cancer cells, or to facilitate transport of MTX across membranes or in vivo barriers. Of the numerous patents issued disclosing methods of using MTX, a variety of patents such as U.S. Pat. Nos. 4,106,488, 4,558,690, and 4,662,359 disclose methods of using MTX to treat cancer. Additionally, U.S. Pat. Nos. 4,396,601 and 4,497,796 describe the use of MTX to select cells that have been transfected with vectors containing a DHFR selectable marker, and U.S. Pat. No. 5,043,270 discloses the use of MTX to select for or assess gene amplification events. The basis for these two latter approaches is that an increase in the number of copies of the DHFR gene within a cell correspondingly increases resistance to MTX.
Despite the broad utility and utilization of MTX, treatment with this agent involves a strong risk to the patient. Since MTX interferes with cell replication and division, actively proliferating non-malignant tissues such as bone marrow and intestinal mucosa are more sensitive to MTX and may demonstrate impaired growth due to treatment. More importantly, MTX is associated with renal and hepatic toxicity when applied in the "high dose regimen" that is typically required for maximum efficiency (Barak et al., J. American Coll. Nutr., 3, 93-96 (1984)). It appears that a major metabolite of MTX, 7-OH-MTX, is the source of this toxicity. In both man and monkeys, MTX is converted in vivo to 7-hydroxymethotrexate (7-OH-MTX) (Borsi et al., Cancer Chemother. Pharmacol., 27, 164-67 (1990); Jacobs et al. J. Clin. Investig., 57, 534-38, (1976)). Also, 7-OH-MTX has been found in both urine and plasma samples of patients following high dose MTX therapy (Watson et al., Cancer Res., 43, 4648 (1983); Breithaupt et al., Cancer Treatment Rep,, 9, 1733 (1982); Heiko et al., Pharmacol., 26, 138-143 (1990); Chatelut et al., J. Pharmaceutical Sci., 80, 730-34 (1991); Lopez et al., Biochemical Pharmacol., 35, 2834-36 (1986)).
To alleviate MTX-induced toxicity, high dose MTX therapy can be administered in conjunction with citrovorum factor as a "rescue" agent for normal cells (Christenson et al., J. Clin. Oncol., 6, 797-801 (1988)). While citrovorum factor rescue reduces MTX toxicity to non-malignant cells, it does not solve the problem of renal and hepatic impairment due to the formation of 7-OH-MTX.
Because of the undisputed value of MTX in therapy and research, attempts have been made to increase the effectiveness of MTX and decrease the problems attendant with its use. Many investigators have modified the structure of MTX in attempts to synthesize more potent MTX derivatives. The most effective derivatives include aminopterin, which possesses a hydrogen instead of a methyl group at position N-10, and 4-amino derivatives with halogen substitution on the para-aminobenzoic moiety, such as dichloromethotrexate (Frei et al., Clin. Pharmacol. and Therap., 6, 160-71 (1965)). Additional MTX derivatives have been synthesized by: (i) preparing ester derivatives of the glutamyl moiety, (ii) replacing the glutamic acid with amino acids and peptides, (iii) adding a methyl group at the 7-position, (iv) poly-(L-lysine) conjugation, and (v) substituting the gamma amides (Rosowsky and Yu, J. Med. Chem., 21, 170-75 (1978); Rosowsky et al., J. Med. Chem., 21, 380-86 (1978); Chaykovsky et al., J. Med. Chem., 18, 909-12 (1975); Rosowsky and Chen, J. Med. Chem., 18, 1308-11 (1974 )). More recent modification attempts include the synthesis of lysine and ornithine derivatives of MTX (Kempton et al., J. Med Chem., 25, 475-477 (1982); Patil et al., J. Med. Chem., 32, 1559-65 (1989)). These attempts to improve the efficacy of MTX have not yet proven entirely successful. Whereas some of the MTX derivatives, like 7-methyl substituted MTX (Rosowsky and Chen, J. Med. Chem., 18, 1308-11 (1974)), demonstrate impaired antifolate antagonism, others, such as 3', 5'-difluoro MTX, demonstrate little or no increase in biological activity as compared with MTX (Tomcuf, J. Organic Chem., 26, 3351 (1961)). Still other derivatives, like the 2' and 3' monoflourinated derivatives of aminopterin, appear promising, but animal studies remain to be performed (Henkin and Washtien, J. Med. Chem., 26, 1193-1196 (1983)). Similarly, 7,8-dihydro-8-methyl-MTX has been prepared, but the biological properties of this and other compounds remain to be fully investigated (Chaykovsky, J. Org. Chem., 40 (1), 145-146 (1975) ).
Consequently, there remains a need for MTX derivatives having improved or at least equivalent efficacy as MTX and having reduced toxicity for normal cells. One study investigated the influence of lipophilicity and carboxyl group content on the ability of MTX derivatives to undergo 7-hydroxylation in vitro (Rosowsky et al., Biochem. Pharmacol., 40, 851-857 (1990)). While increasing lipophilicity was found to facilitate hydroxylation, the addition of two to five poly-glutamyl residues to the MTX molecule caused a decrease in the rate of hydroxylation at the 7-position. However, this study did not determine the effectiveness of the glutamylated derivatives at inhibiting DHFR. Thus, it is an object of the present invention to provide MTX derivatives that compare with MTX in ability to inhibit DHFR and demonstrate reduced hydroxylation at the C-7 position.
In particular, it is an object of the present invention to provide novel analogs of MTX which modulate at least one cellular function, such as DHFR-mediation of DNA synthesis or repair, and show reduced hydroxylation at the 7-position, as compared with MTX. It is an additional object of the present invention to provide pharmaceutical compositions comprising therapeutically acceptable excipients and novel analogs of MTX. It is a further object of the present invention to provide methods of synthesizing such novel analogs of MTX and methods of modulating at least one cellular function using such novel analogs of MTX.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.